Derivatives of alpha-phenylthiocarboxylic and $g(a)-phenyloxy-carboxylic acids useful for the treatment of diseases responding to pparalpha activation

ABSTRACT

Formula (I) compounds are described in which the substituents have the meanings described in the text, and which are useful for the treatment of diseases responding to PPARα activation, such as heart failure, the hyperlipaemias and atherosclerosis.

The invention described herein relates to derivatives ofα-phenylthiocarboxylic and α-phenyloxycarboxylic acids, useful for thetreatment of diseases responding to PPARα activation (PeroxisomeProliferator-Activated Receptor alpha), of general formula (I):

in which:

-   -   R represents —H; —YCR5R6COX; monocyclic, bicyclic or tricyclic        aryl or heteroaryl, possibly substituted by one or more        —YCR5R6COX halogen, nitro, hydroxy, alkyl groups and alkoxy,        possibly substituted by one or more halogen groups; monocyclic,        bicyclic or tricyclic arylalkyl or heteroarylalkyl, in which the        aryl or heteroaryl may possibly be substituted by one or more        —YCR5R6COX halogen, nitro, hydroxy, alkyl groups and alkoxy        possibly substituted by one or more halogen groups; in which the        heteroaryl may possibly be charged, of the type:    -   in which the positive charge is balanced by a suitable negative        counterion;    -   m represents 0-1;    -   n represents 0-3; when n represents 1, R3 and R4, which may be        the same or different, are selected from H or alkyl C₁-C; when n        represents 2 or 3, R3 it is equal to R4 and represents H;    -   p represents 0-1;    -   X represents —OH, —O-alkyl C₁-C₃;    -   R1 and R2, which may be the same or different, are selected        from: —H; alkyl C₁-C₅, -alkoxy, possibly substituted by one or        more halogen groups;    -   -phenoxy, possibly substituted by one or more halogen, nitro,        hydroxy, alkyl groups;    -   -benzyloxy, possibly substituted by one or more halogen, nitro,        hydroxy, alkyl groups;    -   —COX;    -   or R1 or R2 together with COX of general formula (I) form a        cycle of the type:    -   R5 and R6, which may be the same or different, are selected from        the groups listed for R1 and R2;    -   Q and Z, which may be the same or different are selected from:        NH, O, S, —NHC(O)O—, NHC(O)NH—, —NHC(O)S—, —OC(O)NH—, —NHC(S)O—,        —NHC(S)NH—, —C(O)NH—;    -   and Y represents O, S.

The diseases that respond to activation of PPARα according to theinvention described herein are heart failure, the hyperlipaemias andatherosclerosis.

The PPARs, which are members of the superfamily of nuclear receptors,are transcription factors activated by ligands that regulate geneexpression.

Various different isoforms of PPAR have been identified: PPARα, PPARδ(sometimes indicated as β) and PPARγ (J. Med. Chem. 2000, 43, 527-550;Nature 2000, 405, 421-424).

PPARα belongs to the large family of the steroid hormone receptors(Kersten et al., Nature 2000, 405: 421-424).

This receptor was first identified on the basis of its control of thegenes coding for fatty acid oxidation enzymes in response to peroxisomeproliferators such as the derivatives of fibric acid (Issemann andGreen, Nature 1990, 347: 645-650).

Leone et al., in Proc. Natl. Acad. Sci. USA 1999, 96: 7473-7478,confirmed the critical role of fatty acids in tissues played by PPARα.

Heart failure is an important cause of disability and sudden death. Itis due to inability of the heart to pump blood in sufficient amounts tomeet the metabolic needs of the various tissues.

This condition is accompanied by profound changes in the control systemof the electrical and mechanical functions of the heart. The biochemicaland neurohormonal abnormalities observed constitute a mechanism ofadaptation to the altered haemodynamic condition of the decompensatedheart, characterised mainly by a reduction in cardiac output, anincrease in peripheral resistances and retention of blood upstream ofthe failing heart, with consequent atrial dilation and retrogradedecompensation.

The physiopathological mechanisms involved in the onset, development andprogression of heart failure still need to be partly clarified.

Compounds useful for the treatment of diseases responding to PPARαactivation are already known.

In Gen. Pharmacol. September 1995; 26(5):897-904, it is reported thatetomoxir has a beneficial effect on cardiac performance and that thePPARs are involved.

In Prostaglandins Leukot. Essent. Fatty Acids; May-June 1999; 60(5-6):339-43, etomoxir and PPARα are reported to be involved in the control oflipid metabolism.

In Am. J. Physiol. Renal. Physiol. April 2000; 278(4):F667-75 it isreported that etomoxir is a PPARα activator and that this activationinduces a regulation of fatty acid oxidation.

In Circulation 1997, 96:3681-3686, and in Br. J. Pharmacol. 1999,126:501-507, etomoxir is reported to be effective in improvingmyocardial function in animal models of hypertrophy and heart failure.

In Clin. Sci. (Colch) July 2000; 99(1):27-35, it is reported thatpatients with heart failure have improved cardiac functions aftertreatment with etomoxir.

In Curr. Opin. Lipidol. 1999, 10: 245-247, it is reported that, byactivating PPARα, the fibrates stimulate fatty acid oxidation, inhibitinflammation of the vascular walls and protect against atherosclerosis.

In WO 98/05331 it is reported that, by activating PPARα, the fibrateshave a protective effect against hypertension, coronary artery disordersand atheromatous phenomena caused by diabetes.

To date, however, there are still very few compounds available capableof activating PPARα and proving useful for the treatment of cardiacdecompensation.

In this sector of medicine, then, there is a strongly perceived need forincreasingly specific new drugs for the treatment of this condition.

The above-mentioned known compounds are not without certain drawbacks.

In fact, in Therapie September-October 1991; 46(5):351-4, it is reportedthat the fibrates cause several side effects such as skin reactions,haemorrhages, pancreatitis and nervous system disorders.

In Current Pharmaceutical Design, 1998; 4; 1-15, etomoxir is reported toinduce myocardial hypertrophy and increase the risk of myocardialinfarction.

There is therefore a strongly perceived need for new PPARα activatorsendowed with curative activity for the above-mentioned diseaseconditions, but which do not present the drawbacks of theabove-mentioned known compounds.

It has now surprisingly been found that the formula (I) compounds arePPARα activators and that they lend themselves to use in the treatmentof diseases responding to activation of said PPARα.

The diseases responding to PPARα activation, as outlined above, includeheart failure, the hyperlipaemias and atherosclerosis.

The object of the invention described herein consists in formula (I)compounds and their use in the medical field.

A further object of the invention described herein consists inpharmaceutical compositions containing as their active ingredient aformula (I) compound and at least one pharmaceutically acceptableexcipient and/or diluent.

A further object of the invention described herein consists in the useof formula (I) compounds for the preparation of a medicine for thetreatment of diseases responding to PPARα activation, examples of whichare heart failure, the hyperlipaemias and atherosclerosis, though notexclusively these.

The following examples further illustrate the invention.

General Synthetic Methods

The following diagrams illustrate the methods used for the synthesis ofthe formula (I) compounds.

Unless otherwise specified, the meaning of the various symbols coincideswith that indicated in general formula (I). The hydrolysis proceduredescribed in method A can also be applied to the other methods.

The preparation of compounds of general formula (I) was accomplished byreacting the general formula II compound with a base, preferablyinorganic and preferably sodium hydride, to form the correspondinganion, which was then reacted with a general formula III compoundcontaining a leaving group, such as chlorine, bromine, iodine, mesyl,tosyl and diazo (in the case of the diazo group, bivalent rhodiumacetate dimer is used instead of an inorganic base as a catalyst), e.g.2-methyl-alpha-bromoiso-butyrrate, in a polar solvent such asacetonitrile, toluene or preferably dimethylformamide, for a period oftime ranging from 18 to 48 hours at a temperature ranging from 10 to 50°C., preferably 25° C. The product thus obtained was submitted to basicor acid hydrolysis using, for example, NaOH, or, for example, a mixtureof HCl/acetic acid, at a temperature ranging from 10 to 100° C.,preferably 25° C., for a time period ranging from 1 hour to 72 hours,preferably 3 hours, to yield the corresponding acid I A.

The preparation of compounds with general formula (I) was accomplishedstarting from compounds of general structure IV, which were reacted withan alcohol of general structure V in the classic conditions of theMitsunobu reactions, as described in Synthesis 1981, 1-28, usinganhydrous and aprotic solvents such as benzene, toluene, ether orpreferably tetrahydrofuran, for a period of time ranging from 30 minutesto 72 hours, preferably 48 hours, at a temperature ranging from 10 to40° C., preferably 25° C.

The compounds prepared with this method were obtained starting fromgeneral structure VI dissolved in aprotic solvents, e.g. toluene, ether,benzene, but preferably tetrahydrofuran, then added with the relatedisocyanate, thioisocyanate or chloroformiate VII, possibly in thepresence of an inorganic or organic base, preferably triethylamine in acatalytic or stoichiometric amount and leaving the mixture to react fora period of time ranging from 6 to 72 hours, preferably 48 hours at atemperature ranging from 10 to 40° C., preferably 25° C. If K is equalto COOH condensing agents such as diethylphosphoro-cyanidate, EEDQ, DCCor CDI and the like are used in a ratio of 1-3 equivalents to thesubstrates, preferably 1-1.5 equivalents, or one proceeds via theformation of the chloride of the acid, performing the condensationreaction in organic solvents such as DMF, CH₃CN, CHCl₃, THF and thelike, at a temperature ranging from 20 to 80° C., preferably 25° C., ina reaction time ranging from 18 hours to 3 days, preferably 24 hours.

The preparation of general formula compounds (a) (m and n are equal tozero and Y and Q are equal to O and/or S) was accomplished, for example,according to the procedure described in Tetrahedron, 1990, 46 (3),967-978 starting with product IV which was reacted with a generalformula III compound containing a leaving group, such as chlorine,bromine, iodine, mesyl, tosyl and diazo (in the case of the diazo group,bivalent rhodium acetate dimer is used as a catalyst instead of aninorganic base), e.g. 2-methyl-alpha-bromoisobutyrrate, in the presenceof a base, such as potassium carbonate, and of a catalyst for phasetransfer, such as, for example, tetrabutylammonium bromide (TBAB) inaprotic solvents such as toluene, at temperatures ranging from 25° C. tothe reflux temperature of the solvent selected, for a period of timeranging from 1 to 5 days, preferably 2 days.

EXAMPLE 1 Preparation of methyl 2-(4-hydroxyphenylthio)isobutyrate(ST1923)

Method A Step 1

To 4-mercaptophenol (0.500 g, 4.0 mmol) in 10 mL of anhydrous CH₃CN wasadded NaH 80% (0.144 g, 4.8 mmol). The mixture was cooled to 0° C. andmethyl-α-bromoisobutyrate (0.724 g, 4.0 mmol) was added after 5 minutes.The reaction was left at room temperature for two days under magneticstirring. The reaction mixture was then poured into H₂0 and extractedwith ethyl acetate; the aqueous phase was then acidified and extractedagain with ethyl acetate. The pooled organic phases were dried onNa₂SO₄, filtered and evaporated. The residue obtained was purified bysilica gel chromatography using as eluent CHCl₃. 0.760 g of product wereobtained (yield: 84%); Mp (melting point): 110-112° C.; TLC: silica gel,eluent CHCl₃, Fr (frontal ratio): 0.11; ¹H NMR (CDCl₃, 300 MHz) δ 7.30(d, 2H), 6.73 (d, 2H), 5.57 (brm, 1H), 3.70 (s, 3H), 1.45 (s, 6H); HPLC:Column: Symmetry—C₁₈, (5 μm) 4.6×250 mm, R. T. (Room Temperature),mobile phase CH₃CN/H₂O 50/50 (v/v), pH: as it is, flow rate: 0.75mL/min, 205 nm UV detector, retention time 10.14 min; E. A. (elementalanalysis) conforms for C₁₁H₁₄O₃S.

EXAMPLE 2 Preparation of 2-(4-hydroxyphenylthio)isobutyric acid (ST1981)

Method A Step 2

To methyl 2-(4-hydroxyphenylthio)isobutyrate (ST1923) (0.200 g, 0.88mmol) were added 2.7 mL of acetic acid and 2.7 mL of 37% hydrochloricacid and the mixture thus obtained was left overnight refluxing undermagnetic stirring. The solution was then poured into water and theaqueous phase extracted with ethyl acetate. The organic phase was thendried on Na₂SO₄, filtered and evaporated. 0.161 g of product wereobtained (yield: 87%); Mp 152-154° C.; TLC: silica gel, eluentCHCl₃/CH₃OH 9/1, Fr: 0.38; ¹H NMR (DMSO, 300 MHz) δ 7.23 (d, 2H), 6.72(d, 2H), 3.30 (brm, 2H), 1.30 (s, 6H); HPLC: Column: Inertisil ODS—3 (5μm) 4.6×250 mm, R. T., mobile phase CH₃CN/KH₂PO₄ 50 mM 40/60 (v/v), pH:as it is, flow rate: 0.75 mL/min, 205 nm UV detector, retention time7.39 min; KF: 0.5% H₂O; E. A. conforms for C₁₀H₁₂O₃S.

EXAMPLE 3 Preparation of methyl 2-(3-hydroxyphenylthio)isobutyrate(ST2047)

The product was prepared according to the procedure described in methodA (step 1), starting from 3-mercaptophenol (2.000 g, 15.9 mmol) in 40 mLof anhydrous CH₃CN, 80% NaH (0.572 g 19.1 mmol) at 0° C. After 5 minutesmethyl-2-bromoisobutyrate (2.88 g, 15.9 mmol) was added to thesuspension. The reaction mixture thus obtained was left overnight undermagnetic stirring at room temperature. The reaction mixture was thenpoured into H₂O and extracted with ethyl acetate. The organic phase wasdried on anhydrous sodium sulphate, filtered and evaporated. The residueobtained was purified by silica gel chromatography using as eluentCHCl₃/CH₃OH 98/2. 2,900 g of product were obtained (yield: 81%); Mp:41.5-42.5° C.; TLC: silica gel, eluent CHCl₃/CH₃OH 98/2, Fr: 0.23; ¹HNMR (CDCl₃, 300 MHz) δ 7.19 (t, 1H), 7.00 (d, 1H), 6.95 (brt, 1H), 6.81(dd, 1H), 3.69 (s, 3H), 1.50 (s, 6H); HPLC: Column: Inertisil ODS—3 (5μm) 4.6×250 mm, R.T., mobile phase CH₃CN/H₂O 50/50 (v/v), pH: as it is,flow rate: 0.75 mL/min, 205 nm WV detector, retention time 13.82 min;KF: 0.3% H₂O; E. A. conforms for C₁₁H₁₄O₃S.

EXAMPLE 4 Preparation of methyl2-[4-[2-(4-chlorophenyl)ethoxy]phenyl-thio]isobutyrate (ST1929)

Method B

To methyl 2-(4-hydroxyphenylthio)isobutyrate (ST1923, prepared asdescribed in example 1) (0.800 g, 3.54 mmol) and 4-chlorophenethylalcohol (0.554 g, 3.54 mmol) in 20 mL of anhydrous THF were added DEAD(0.801 g, 4.6 mmol) and triphenylphosphine (1.205 g, 4.6 mmol) in smallportions, keeping the temperature below 30° C. The reaction mixture wasleft overnight under magnetic stirring at room temperature. The solventwas then evaporated and the residue purified by silica gelchromatography using as eluent hexane/ethyl acetate 9/1. 0.416 g of oilyproduct were obtained (yield: 32%); TLC: silica gel, eluent hexane/ethylacetate 9/1, Fr: 0.32; ¹H NMR (CDCl₃, 300 MHz) δ 7.40-7.19 (m, 6H), 6.80(d, 2H), 4.15 (t, 2H), 3.65 (s, 3H), 3.08 (t, 2H) 1.45 (s, 6H); HPLC:Column: Symmetry—C₁₈, (5 μm) 4.6×250 mm, R. T. , mobile phase CH₃CN/H₂O70/30 (v/v), pH: as it is, flow rate: 0.75 mL/min, 205 nm UV detector,retention time 31.40 min; KF: 0.4% H₂O; E. A. conforms for C₁₉H₂₁ClO₃S.

EXAMPLE 5 Preparation of methyl2-[4-[2-(1-indolyl)ethoxy]phenyl-thio]isobutyrate (ST1983) Preparationof the intermediate product 1-(2-hydroxy-ethyl)indole

The intermediate product, reported in J. Med. Chem. 1998, 41/10,1619-1639, was prepared according to the procedure described therein,except for the duration of the reaction time (30 hours rather than 30minutes), starting from indole (5.0 g, 42.7 mmol), KOH (3.6 g, 64.1mmol) and bromoethanol (6.4 g, 51.3 mmol) in 50 ml of anhydrous DMSO, atT: 25-30° C., to obtain 5 g of oily product (yield: 73%).

Preparation of methyl 2-[4-[2-(1-indolyl)ethoxy]phenylthio]isobutyrate(ST1983)

The product was prepared according to the procedure described in methodB starting from methyl 2-(4-hydroxyphenylthio)isobutyrate (ST1923,prepared as described in example 1) (0.671 g, 2.97 mmol),1-(2-hydroxyethyl)indole (0.478 g, 2.97 mmol), DEAD (0.672 g, 3.86 mmol)and triphenylphosphine (1.011 g, 3.86 mmol) added in small portionskeeping the temperature below 30° C., in 15 mL of anhydrous THF. Thereaction mixture was left under magnetic stirring for 48 hours at roomtemperature. Then the solvent was evaporated and the residue purified bysilica gel chromatography using hexane/ethyl acetate 8/2 as eluent. Atotal of 0.500 g of still impure product was obtained which wasdissolved in ethyl acetate and washed with a solution of NaOH 1N. Theorganic phase was dried and evaporated to yield a residue of 0.230 gwhich was further purified by silica gel chromatography using as eluentCHCl₃. 0.184 g of oily product were obtained (yield: 17%); TLC: silicagel, eluent hexane/ethyl acetate 8/2, Fr: 0.29; ¹H NMR (CDCl₃, 300 MHz)δ 7.62 (d, 1H), 7.40-7.10 (m, 6H), 6.78 (d, 2H), 6.50 (d, 1H), 4.50 (m,2H), 4.24 (m, 2H), 3.61 (s, 3H), 1.40 (s, 6H); HPLC: Column:Symmetry—C₁₈, (3.5 μm) 4.6×75 mm, R. T., mobile phase CH₃CN/H₂O 60/40(v/v), pH: as it is, flow rate: 0,90 mL/min, 205 nm UV detector,retention time 10.70 min; KF: 1.7% H₂O; E. A. conforms for C₂₁H₂₃NO₃S.

EXAMPLE 6 Preparation of methyl2-[4-[2-(2-naphthyl)ethoxy]phenyl-thio]isobutyrate (ST2011)

The product was prepared according to the procedure described in methodB starting from methyl 2-(4-hydroxyphenylthio)isobutyrate (ST1923,prepared as described in example 1) (1.000 g, 4.42 mmol),2-(2-naphthyl)ethanol (0.760 g, 4.42 mmol), DEAD (1.000 g, 5.75 mmol)and triphenylphosphine (1.500 g, 5.75 mmol) added in small portionskeeping the temperature below 30° C., in 30 mL of anhydrous THF. Thereaction mixture was left overnight under magnetic stirring at roomtemperature. The solvent was then evaporated and the residue purified bysilica gel chromatography using as eluent hexane/ethyl acetate 9/1.1.262 g of product were obtained (yield: 75%); Mp: 56-57° C.; TLC:silica gel, eluent hexane/ethyl acetate 9/1, Fr: 0.23; ¹H NMR (CDCl₃,300 MHz) δ 7.85-7.70 (m, 4H), 7.45-7.28 (m, 5H), 6.83 (d, 2H), 4.27 (t,2H), 3.65 (s, 3H), 3.26 (t, 2H), 1.45 (s, 6H); HPLC: Column: InertisilODS—3 (5 μm) 4.6×250 mm, R. T., mobile phase CH₃CN/H₂O 80/20 (v/v), pH:as it is, flow rate 0.75 mL/min, 205 nm UV detector, retention time23.51 min; KF: 0.16% H₂O; E. A. conforms for C₂₃H₂₄O₃S.

EXAMPLE 7 Preparation of2-[4-[2-(2-naphthyl)ethoxy]phenylthio]isobutyric acid (ST2036)

To a solution of ST2011 (prepared as described in example 6) (0.489 g,1.29 mmol) in 30 mL of methanol were added 12.9 mL of NaOH 1N. Thesolution thus obtained was left to reflux overnight. The solution wasthen cooled, diluted with water and acidified, and the aqueous phase wasextracted with ethyl acetate. The organic phase was dried over anhydrousNa₂SO₄, then evaporated in vacuo and the residue purified by silica gelchromatography using as eluent chloroform. 0.360 g of product wereobtained (yield: 76.2%); Mp: 103-104° C.; TLC: silica gel, eluentCHCl₃/CH₃OH 98/2, Fr: 0.13; ¹H NMR (CDCl₃, 300 MHz) δ 7.80 (m, 3H), 7.70(s, 1H), 7.50-7.38 (m, 5H), 6.83 (d, 2H), 4.26 (t, 2H), 3.35 (t, 2H),1.48 (s, 6H); HPLC: Column: Inertisil ODS—3 (5 μm) 4.6×250 mm, R. T.,mobile phase CH₃CN/KH₂PO₄ 75/25 (v/v), pH: as it is, flow rate: 0.75mL/min, 205 nm UV detector, retention time 13.07 min; KF: 1% H₂O; E. A.conforms for C₂₂H₂₂O₃S.

EXAMPLE 8 Preparation of methyl2-[4-[[(4-methoxybenzyl)carbamoyl]oxy]phenylthio]isobutyrate (ST2031)

Method C

To ST1923 (0.482 g, 2.13 mmol) (prepared as described in example 1) in10 mL of anhydrous THF were added p-methoxybenzylisocyanate (0.417 g,2.56 mmol) and 0.010 g of triethylamine. The solution was left undermagnetic stirring at room temperature for 48 hours. After this timeperiod the solvent was evaporated and the residue purified by silica gelchromatography using as eluent CHCl₃/CH₃OH 98/2. 0.410 g of product wereobtained (yield: 50%); Mp: 64-65° C.; TLC: silica gel, eluent CHCl₃, Fr:0.14; ¹H NMR (CDCl₃, 300 MHz) δ 7.44 (d, 2H), 7.28 (d, 2H), 7.10 (d,2H), 6.90 (d, 2H), 5.29 (brm, 1H), 4.39 (d, 2H), 3.80 (s, 3H), 3.65 (s,3H), 1.48 (s, 6H); HPLC: Column: Inertisil ODS—3 (5 μm) 4.6×250 mm, R.T., mobile phase CH₃CN/H₂O 70/30 (v/v), pH: as it is, flow rate 0.75mL/min, 205 nm UV detector, retention time 11.22 min; E. A. conforms forC₂₀H₂₃NO₅S.

EXAMPLE 9 Preparation of methyl2-[3-[[(4-methoxy-benzyl)carbamoyl]oxy]phenylthio]isobutyrate (ST2139)

The product was prepared according to the procedure described in methodC starting from ST2047 (prepared as described in example 3) (0.240 g,1.06 mmol) in 7 mL of anhydrous THF, p-methoxybenzylisocyanate (0.207 g,1.27 mmol) and 0.010 g of triethylamine, leaving the solution to stirfor 18 hours at room temperature. Then 0.086 g (0.53 mmol) ofp-methoxybenzylisocyanate were added and the mixture was left undermagnetic stirring for additional 6 hours at room temperature. Thesolvent was then evaporated to dryness and the residue purified bysilica gel chromatography using as eluent hexane/ethyl acetate 7/3.0.320 g of product were obtained which were further purified by washingwith Na₂CO₃. 0.200 g of oily product were obtained (yield 48%); TLC:silica gel, eluent hexane/ethyl acetate 7/3, Fr: 0.37; ¹H NMR (CDCl₃,300 MHz) δ 7.35-7.18 (m, 6H), 6.90 (d, 2H), 5.25 (brm, 1H), 4.40 (d,2H), 3.80 (s, 3H), 3.62 (s, 3H), 1.50 (s, 6H); HPLC: Column: InertisilODS—3 (5 μm) 4.6×250 mm, R. T., mobile phase CH₃CN/H₂O 50/50 (v/v), pH:as it is, flow rate: 0.75 mL/min, 205 nm UV detector, retention time47.02 min; E. A. conforms for C₂₀H₂₃NO₅S.

EXAMPLE 10 Preparation of methyl2-[4-(2-methoxy-1,1-dimethyl-2-oxoethoxy)phenylthio]isobutyrate (ST1982)

Method D

To methyl 2-(4-hydroxyphenylthio)isobutyrate (ST1923, prepared asdescribed in example 1) (0.250 g, 1.11 mmol) in 15 mL of anhydroustoluene, were added K₂CO₃ (0.306 g, 2.22 mmol) and tetrabutylammoniumbromide (TBAB) (0.0193 g, 0.06 mmol); the mixture was heated at 100° C.and after 5 minutes methyl-2-bromoisobutyrrate (0.803 g, 4.44 mmol) wasadded. The reaction mixture was then left refluxing for two days (oilbath temperature 130° C.). Then the mixture was filtered and the solidwashed with toluene. The pooled organic phases were dried and the oilyresidue was dissolved with ethyl acetate and washed with NaOH 1N. Theresidue obtained after evaporation of the organic solvent was purifiedby silica gel chromatography using as eluent hexane/ethyl acetate 9/1.0.145 g of oily product were obtained (yield: 40%); TLC: silica gel,eluent hexane/ethyl acetate 9/ 1, Fr: 0.17; ¹H NMR (CDCl₃, 300 MHz) δ7.31 (d, 2H), 6.74 (d, 2H), 3.75 (s, 3H), 3.65 (s, 3H), 1.60 (s, 6H),1.45 (s, 6H); HPLC: Column: Symmetry—C₁₈, (3.5 μm) 4.6×75 mm, R. T.,mobile phase CH₃CN/H₂O 50/50 (v/v), pH: as it is, flow rate: 0.75mL/min, 205 nm UV detector, retention time 13.00 min; E. A. conforms forC₁₆H₂₂O₅S.

EXAMPLE 11 Preparation of methyl2-[3-[2-(3-hydroxy-phenoxy)ethoxy]phenoxy]isobutyrate (ST1877) andmethyl2-[3-[2-[3-(2-methoxy-1,1-dimethyl-2-oxoethoxy)phenoxy]ethoxy]phenoxy]isobutyrate(ST1878)

The products were prepared according to the procedure described inmethod D starting from 3,3-ethylenedioxidephenol (2.000 g, 8.1 mmol),K₂CO₃ (4.500 g, 32.4 mmol), TBAB (0.131 g, 0.4 mmol) andmethyl-2-bromoisobutyrate (11.611 g, 64 mmol) in 100 mL of toluene. Thereaction mixture was heated at 130° C. for three days, then cooled andfiltered. The solid obtained was washed with toluene, the pooled organicphases were evaporated to dryness in vacuo and the oily residue waspurified by silica gel chromatography using as eluent hexane/ethylacetate 8/2. Two products were obtained: the monoderivative ST1877(0.700 g) (yield: 25%) and the bisderivative ST1878 (1.100 g) (yield:30.4%).

Analytical Data for ST1877

Melting point: 77-79° C.; ¹H NMR (CDCl₃, 300 MHz) δ 7.13 (t, 2H),6.62-6.40 (m, 6H), 4.25 (s, 4H), 3.78 (s, 3H) 1.60 (s, 6H); HPLC: ColumnInertisil ODS—3 (5 μm); 4.6×250 mm, R. T.; mobile phase: CH₃CN/H₂O(60/40 v/v), pH: 3.2, flow rate: 1.0 mL/min, 205 nm UV detector,retention time: 8.76 min; E. A. conforms for C₁₉H₂₂O₆.

Analytical Data for ST1878

Melting point: 60-62° C.; ¹H NMR (CDCl₃, 300 MHz) δ 7.13 (t, 2H), 6.60(d, 2H), 6.41 (m, 4H), 4.26 (s, 4H), 3.78 (s, 6H) 1.60 (s, 12H); HPLC:Column Inertisil ODS—3 (5 μm), 4.6×250 mm, R. T., mobile phase:CH₃CN/H₂O (60/40 v/v), pH: 3,2, flow rate: 1.0 mL/min, 205 nm UVdetector, retention time: 23.92 min; E. A. conforms for C₂₄H₃₀O₈.

EXAMPLE 12 Preparation of dimethyl2-[4-[1-(4-hydroxyphenyl)-1-methyl-ethyl]phenoxy]malonate (ST2020)

The product was prepared as described for method A, step 1 according tothe following procedure: to a suspension of bivalent rhodium acetatedimer (0.220 g, 0.5 mmol) and bisphenol A(2,2-bis-(4-hydroxyphenyl)-propane) (3.400 g, 15 mmol) in 100 mL ofanhydrous toluene, was added drop-wise, under nitrogen flow, a solutionof diazomalonate (2.846 g, 18 mmol) (prepared as described in Org.Synth.: 1973, V, 179) in 50 mL of anhydrous toluene, taking care to keepthe temperature between 15 and 20° C. The reaction mixture was thenrefluxed at 120-130° C. for 24 hours under nitrogen. Then the reactionmixture was filtered and the toluene evaporated in vacuo. The residueobtained was purified by silica gel chromatography using as eluenthexane/ethyl acetate 8/2. 1.700 g of oily product were obtained (yield:32%); TLC: silica gel, eluent hexane/ethyl acetate 7/3, Fr. 0.23; ¹H NMR(CDCl₃, 300 MHz) δ 7.16 (m, 4H), 6.90 (d, 2H), 6.87 (d, 2H), 5.12 (s,1H), 3.90 (s, 6H), 1.62 (s, 6H); HPLC: Column: Inertisil ODS—3 (5 μm)4.6×250 mm, R. T., mobile phase CH₃CN/H₂O 70/30 (v/v), pH: as it is,flow rate: 0.75 mL/min, 205 nm UV detector, retention time 7.00 min; KF:0.6% H₂O; E. A. conforms for C₂₀H₂₂O₆.

EXAMPLE 13 Preparation of dimethyl2-[4-(1-{4-[2-methoxy-1-(methoxy-carbonyl)-2-oxoethoxy]phenyl}-1-methylethyl)phenoxy]malonate(ST2048)

The product was prepared as described for method A, step 1, according tothe procedure already described in example 12 starting from bivalentrhodium acetate dimer (0.0885 g, 0.2 mmol) and ST2020 (1.230 g, 3.4mmol) (prepared as described in example 12) in 36 mL of anhydroustoluene, adding diazomalonate (1.882 g, 11.9 mmol) dropwise in 18 mL ofanhydrous toluene, taking care to keep the temperature between 15 and20° C. The reaction mixture was refluxed at 120-130° C. for 24 hoursunder nitrogen. Then the reaction mixture was filtered and the toluenewas evaporated in vacuo. The residue obtained was purified by silica gelchromatography using as eluent hexane/ethyl acetate 8/2. 0.430 g of oilyproduct were obtained (yield: 26%); TLC: silica gel, eluent hexane/ethylacetate 6/4, Fr: 0.46; ¹H NMR (CDCl₃, 300 MHz) δ 7.20 (d, 4H), 6.90 (d,4H), 5.22 (s, 2H), 3.90 (s, 12H), 1.61 (s, 6H); HPLC: Column: InertisilODS—3 (5 μm) 4.6×250 mm, R: T., mobile phase CH₃CN/H₂O 70/30 (v/v), pH:as it is, flow rate: 0.75 mL/min, 205 nm UV detector, retention time9.68 min; KF: 0.7% H₂O; E. A. conforms for C₂₅H₂₈O₁₀.

EXAMPLE 14 Preparation of methyl2-[3-[2-(2-naphthyl)ethoxy]phenyl-thio]isobutyrate (ST2167)

The product was prepared according to the procedure described in methodB (with exception of DEAD which was replaced by DIAD) starting frommethyl 2-(3-hydroxyphenylthio)isobutyrate (ST2047) (1.110 g, 4.9 mmol),2-(2-naphthyl)ethanol (0.842 g, 4.9 mmol), DIAD (1.290 g, 6.37 mmol),and triphenylphosphine (1.670 g, 6.37 mmol) in 20 mL of anhydrous THF.The reaction mixture was left overnight under magnetic stirring at roomtemperature. Then the solvent was removed under vacuum and the residuepurified by silica gel chromatography using as eluent hexane/ethylacetate 7/3. The product was further purified by dissolving it in ethylacetate and washing the organic phase with a solution of Na₂CO₃. Theorganic phase was then dried on sodium sulphate anhydrous, filtered andthe solvent was evaporated in vacuo. 1.14 g of product were obtained(yield: 61.2%); TLC: silica gel, eluent hexane/ethyl acetate 9/1, Fr:0.20; ¹H NMR (CDCl₃, 300 MHz) δ 7.80 (m, 3H), 7.75 (s, 1H), 7.45 (m,3H), 7.25 (t, 1H), 7.05 (m, 2H), 6.90 (d, 1H), 4.25 (t, 2H), 3.65 (s,3H), 3.30 (t, 2H), 1.50 (s, 6H); HPLC: Column: Inertisil ODS—3 (5 μm)4.6×250 mm, R. T., mobile phase CH₃CN/H₂O 80/20 (v/v), pH: as it is,flow rate: 0,9 mL/min, 205 nm UV detector, retention time 18.91 min; KF:1.0% H₂O; E. A. conforms for C₂₃H₂₄O₃S.

EXAMPLE 15 Preparation of methyl2-[3-[[[4-(trifluoro-methyl)phenyl]carbamoyl]oxy]phenylthio]isobutyrate(ST2208)

The product was prepared according to the procedure described in methodC starting from ST2047 (0.800 g, 3.54 mmol) (prepared as described inexample 3) in 10 mL of anhydrous THF, 4-trifluoromethylisocyanate (0.749g, 4.25 mmol) and 0.010 g of triethylamine; the reaction time was 18hours instead of 48 hours, at room temperature. The solvent was thenevaporated to dryness and the residue purified by silica gelchromatography using as eluent CHCl₃ and CHCl₃/MeOH 98/2. 0.881 ofproduct were obtained (yield=60%); Mp =66-67° C.; TLC: silica gel,eluent CHCl₃, Fr: 0.38; ¹H NMR (CDCl₃, 300 MHz) δ 7.60 (m, 4H), 7.38 (m,3H), 7.15 (m, 1H), 7.06 (brs, 1H), 3.70 (s, 3H), 1.55 (s, 6H); HPLC:Column: Inertisil ODS—3 (5 μm) 4.6×250 mm, R. T., mobile phaseCH₃CN/KH₂PO₄ 50 mM (60/40 v/v), pH: 3.0 (H₃PO₄ 85%), flow rate: 1mL/min, 205 nm UV detector, retention time 25.46 min; KF: 2.5% H₂O; E.A. conforms for C₁₉H₁₈F₃NO₄S.

EXAMPLE 16 Preparation of methyl2-[4-[[[4-(trifluoro-methyl)phenyl]carbamoyl]oxy]phenylthio]isobutyrate(ST2209)

The title product was prepared according to the procedure described inmethod C starting from ST1923 (0.300 g, 1.33 mmol) (prepared asdescribed in example 1) in 7 mL of anhydrous THF,4-trifluoromethylisocyanate (0.298 g, 1.6 mmol) and 0.010 g oftriethylamine; the reaction time was 18 hours instead of 48 hours, atroom temperature. The solvent was then evaporated to dryness and theresidue purified by silica gel chromatography using as eluenthexane/AcOEt 7/3. 0.340 g of product were obtained (yield: 62%);Mp=110-111° C.; TLC: silica gel, eluent CHCl₃, Fr: 0.34; ¹H NMR (CDCl₃,300 MHz) δ 7.55 (m, 4H), 7.48 (d, 2H), 7.15 (d, 2H), 7.10 (brs, 1H),3.70 (s, 3H), 1.55 (s, 6H); HPLC: Column: Inertisil ODS—3 (5 μm) 4.6×250mm, R. T., mobile phase CH₃CN/KH₂PO₄ 50 mM (60/40 v/v), pH: 3.0 (H₃PO₄85%), flow rate: 1 mL/min, 205 nm WV detector, retention time 25.60 min;E. A. conforms for C₁₉H₁₈F₃NO₄S.

EXAMPLE 17 Preparation of methyl2-[3-[2-(4-chlorophenyl)ethoxy]phenyl-thio]isobutyrate (ST2195)

The title product was prepared according to the procedure described inmethod B starting from methyl 2-(3-hydroxyphenylthio)isobutyrate(ST2047, prepared as described in example 3) (1.00 g, 4.42 mmol), and4-chlorophenethyl alcohol (0.692 g, 4.42 mmol) in 15 mL of anhydrousTHF, to which were added in small portions DIAD (1.16 g, 5.75 mmol) andtriphenylphosphine (1.500 g, 5.75 mmol) keeping the temperature below30° C. The reaction was left overnight under magnetic stirring at roomtemperature. After this period the solvent was evaporated and theresidue was purified by silica gel chromatography using as eluenthexane/AcOEt 9/1. 1.146 g of oily product were obtained (yield: 71%);TLC: silica gel, eluent hexane/AcOEt 9/1, Fr: 0.28; ¹H NMR (CDCl₃, 300MHz) δ 7.25 (m, 6H), 7.00 (m, 1H), 6.90 (d, 1H), 4.15 (t, 2H), 3.65 (s,3H), 3.08 (t, 2H), 1.55 (s, 6H); HPLC: Column: Inertisil ODS 3 (5 μm)4.6×250 mm, R. T., mobile phase CH₃CN/H₂O 80/20 (v/v), pH: as it is,flow rate: 0.75 mL/min, 205 nm UV detector, retention time 19.34 min;KF: 1.7% H₂O; E. A. conforms for C₁₉H₂₁ClO₃S.

EXAMPLE 18 Preparation of methyl2-[3-[2-(1-indolyl)ethoxy]phenyl-thio]isobutyrate (ST2394)

The title product was prepared according to the procedure described inmethod B starting from methyl 2-(3-hydroxyphenylthio)isobutyrate(ST2047, prepared as described in example 3) (1.00 g, 4.42 mmol), and1-(2-hydroxyethyl) indole (prepared as described in example 5) (0.711 g,4.42 mmol) in 20 mL of anhydrous THF, to which were added in smallportions DIAD (1.16 g, 5.75 mmol) and triphenylphosphine (1.500 g, 5.75mmol) keeping the temperature below 30° C. The reaction was leftovernight under magnetic stirring at room temperature. Then the solventwas evaporated to dryness and the residue purified by silica gelchromatography using as eluent hexane/AcOEt 8/2. 0.581 g of oily productwere obtained (yield: 35%); TLC: silica gel, eluent hexane/AcOEt 9/1,Fr: 0.22; ¹H NMR (CDCl₃, 300 MHz) δ: 7.62 (d, 1H), 7.42 (d, 1H),7.30-6.80 (m, 7H), 6.52 (d, 1H), 4.55 (m, 2H), 4.30 (m, 2H), 3.61 (s,3H), 1.50 (s, 6H); HPLC: Column: Supelco—C₁₈ (5 μm) 4.6×150 mm, R. T.,mobile phase CH₃CN/H₂O 70/30 (v/v), pH: as it is, flow rate: 0.90mL/min, 205 nm UV detector, retention time 6.36 min; E. A. conforms forC₂₁H₂₃NO₃S.

EXAMPLE 19 Preparation of methyl2-[3-[(1-methyl-1-methoxy-carbonyl)ethyloxy]phenylthio]isobutyrate(ST2418)

The title product was prepared according to the procedure described inmethod D starting from 2-(3-hydroxyphenyl-thio)isobutyrate (ST2047,prepared as described in example 3) (0.870 g, 3.85 mmol), in 100 mL oftoluene, K₂CO₃ (1.06 g, 7.7 mmol), TBAB (0.062 g, 0.19 mmol) andmethyl-2-bromoisobutyrate (2.8 g, 15.4 mmol). The reaction mixture washeated at 130° C. for three days, then cooled and filtered. The solidobtained was washed with toluene, the pooled organic layers wereevaporated to dryness in vacuo and the oily residue was purified bysilica gel chromatography using hexane/AcOEt 9:1 as the eluent. 1.0 g ofoily product was obtained (yield: 79%); TLC: silica gel, eluenthexane/AcOEt 9/1, Fr: 0.20; ¹H NMR (CDCl₃, 300 MHz) δ: 7.20 (m, 1H),7.05 (d, 1H), 6.95 (s, 1H), 6.90 (d, 1H), 3.80 (s, 3H), 3.65 (s, 3H),1.60 (s, 6H), 1.45 (s, 6H); HPLC: Column: Symmetry—C₁₈ (5 μm) 4.6×150mm, R. T., mobile phase CH₃CN/H₂O 60/40 (v/v), pH: as it is, flow rate:0.75 mL/min, 205 nm UV detector, retention time 9.53 min; E. A. conformsfor C₁₆H₂₂O₅S.

EXAMPLE 20 Preparation of2-[4-[2-(4-chloro-phenyl)ethoxy]phenylthio]-2-methylpropanoic acid (ST2505)

The title product was prepared according to the procedure described ingeneral method A, step 2 starting from a solution of ST1929 (prepared asdescribed in example 4) (0.572 g, 1.57 mmol), in 36 mL of methanol towhich were added 15.7 mL of NaOH 1N. The solution thus obtained wasrefluxed overnight. The solution was then cooled, diluted with water andacidified and the aqueous phase was extracted with AcOEt. The organicphase was evaporated in vacuo and the residue purified by silica gelchromatography using as eluent hexane/AcOEt 7/3. 0.448 g of product wereobtained (yield: 81%); Mp=87-88° C.; TLC: silica gel, eluenthexane/AcOEt 6/4, Fr: 0.30; ¹H NMR (CDCl₃, 300 MHz) δ 7.45 (d, 2H), 7.15(m, 4H), 6.85 (d, 2H), 4.15 (t, 2H), 3.05 (t, 2H), 1.50 (s, 6H); HPLC:Column: Symmetry—C₁₈ (5 μm) 4.6×250 mm, R. T., mobile phaseCH₃CN/ammonium acetate 10 mM 45/55 (v/v), pH: as it is, flow rate: 0.70mL/min, 205 nm UV detector, retention time 4.73 min; E. A. conforms forC₁₈H₁₉ClO₃S.

EXAMPLE 21 Preparation of methyl2-[3-[5-(4-nitrophenyl)furfuryl-oxy]phenylthio]isobutyrate (ST2501)

The title product was prepared according to the procedure described inmethod B starting from methyl 2-(3-hydroxyphenylthio)isobutyrate(ST2047, prepared as described in example 3) (1.02 g, 4.5 mmol) and5-(nitrophenyl)furfuryl alcohol (0.986 g, 4.5 mmol) in 23 mL ofanhydrous THF to which were added in small portions DIAD (1.18 g, 5.85mmol) and triphenylphosphine (1.53 g, 5.85 mmol) keeping the temperaturebelow 30° C. The reaction was left overnight under magnetic stirring atroom temperature. Then the solvent was evaporated and the residuepurified by silica gel chromatography using as eluent hexane/AcOEt9.4/0.6. 0.300 g of product were obtained (yield: 16%); Mp: 81-82° C.;TLC: silica gel, eluent hexane/AcOEt 7/3, Fr: 0.45; ¹H NMR (CDCl₃, 300MHz) δ 8.25 (d, 2H), 7.80 (d, 2H), 7.30 (m, 1H), 7.05 (m, 1H), 7.03 (m,1H), 7.01 (m, 1H), 6.90 (d, 1H), 6.60 (d, 1H), 5.10 (s, 2H), 3.70 (s,3H), 1.50 (s, 6H); HPLC: Column: Symmetry—C₁₈ (5 μm) 4.6×250 mm, R. T.,mobile phase CH₃CN/H2O 85/15 (v/v), pH: as it is, flow rate: 0.85mL/min, 205 nm UV detector, retention time 6.24 min; E. A. conforms forC₂₂H₂₁NO₆S.

EXAMPLE 22 Preparation of2-[3-[2-(4-chlorophenyl)ethoxy]phenylthio]-2-methylpropanoic acid(ST2518)

The title product was prepared according to the procedure described ingeneral method A, step 2 starting from a solution of ST2195 (prepared asdescribed in example 17) (0.150 g, 0.41 mmol) in 9 mL of methanol towhich were added 4 mL of NaOH 1N. The solution thus obtained was leftunder magnetic stirring for 48 hours at room temperature Then thesolution was diluted with water, acidified and the aqueous phase wasextracted with AcOEt. The organic phase was dried on anhydrous Na₂SO₄and filtered, and the solvent was evaporated in vacuo. 0.128 g ofproduct were obtained (yield=88%); Mp: 105-106° C.; TLC: silica gel,eluent CHCl₃/CH₃OH 9.4/0.6, Fr: 0.42; ¹H NMR (CDCl₃, 300 MHz) δ 7.45 (m,5H), 7.10 (m, 2H), 6.80 (dd, 1H), 4.15 (t, 2H), 3.05 (t, 2H), 1.50 (s,6H); HPLC: Column: Symmetry—C₁₈ (5 μm) 4.6×250 mm, R. T., mobile phaseCH₃CN/ammonium acetate 10 mM 35/65 (v/v), pH: as it is, flow rate:0.80mL/min, 205 nm UV detector, retention time 4.66 min; E. A. conforms forC₁₈H₁₉ClO₃S.

EXAMPLE 23 Preparation of methyl2-[4-(2-(2,4-dichloro-phenyl)ethoxy)phenylthio]isobutyrate (ST2531)

The title product was prepared according to the procedure described inmethod B starting from methyl 2-(4-hydroxyphenylthio)isobutyrate(ST1923, prepared as described in example 1) (0.280 g, 1.24 mmol) andDIAD (0.325 g, 1.61 mmol) dissolved in 3 mL of anhydrous THF and addeddrop-wise to a solution of 2,4-dichlorophenethylalcohol (0.260 g, 1.36mmol) and triphenylphosphine (0.422 g, 1.61 mmol) in 4 mL of anhydrousTHF at 0° C. The reaction mixture was left overnight under magneticstirring at room temperature. Then the solvent was evaporated and theresidue purified by silica gel chromatography using as eluenthexane/AcOEt 9.6/0.4. 0.346 g of product were obtained (yield: 70%); Mp:73-74° C.; TLC: silica gel, eluent hexane/AcOEt 9/1, Fr: 0.26; ¹H NMR(CDCl₃, 300 MHz) δ 7.35 (m, 3H), 7.22 (m, 2H), 6.83 (d, 2H), 4.18 (t,2H), 3.65 (s, 3H), 3.20 (t, 2H), 1.45 (s, 6H); HPLC: Column: InertisilODS—3 (5 μm) 4.6×250 mm, R. T., mobile phase CH₃CN/H₂O 85/15 (v/v), pH:as it is, flow rate: 1 mL/min, 205 nm UV detector, retention time 12.58min; KF: 0.4% H₂O; E.A. conforms for C₁₉H₂₀Cl₂O₃S.

EXAMPLE 24 Preparation of methyl2-[3-(2-(2,4-dichloro-phenyl)ethoxy)phenylthio]isobutyrate (ST2534)

The title product was prepared according to the procedure described inmethod B starting from methyl 2-(3-hydroxyphenylthio)isobutyrate(ST2047, prepared as described in example 3) (0.280 g, 1.24 mmol) andDIAD (0.325 g, 1.61 mmol) dissolved in 3 mL of anhydrous THF and addeddrop-wise to a solution of 2,4-dichlorophenethylalcohol (0.260 g, 1.36mmol) and triphenylphosphine (0.422 g, 1.61 mmol) in 4 mL of anhydrousTHF at 0° C. The reaction was left overnight under magnetic stirring atroom temperature. The solvent was then evaporated and the residuepurified by silica gel chromatography using as eluent hexane/AcOEt9.6/0.4. 0.327 g of oily product were obtained (yield: 66%); TLC: silicagel, eluent hexane/AcOEt 9/1, Fr: 0.34; ¹H NMR (CDCl₃, 300 MHz) δ 7.40(d, 1H), 7.20 (m, 3H), 7.00 (m, 2H), 6.90 (dd, 1H), 4.15 (t, 2H), 3.65(s, 3H), 3.20 (t, 2H), 1.45 (s, 6H); HPLC: Column: Inertisil ODS—3 (5μm) 4.6×250 mm, R. T., mobile phase CH₃CN/H₂O 90/10 (v/v), pH: as it is,flow rate: 0.8 mL/min, 205 nm UV detector, retention time: 12.40 min;KF: 0.2% H₂O; E. A. conforms for C₁₉H₂₀Cl₂O₃S.

EXAMPLE 25 Preparation of methyl2-[3-(2-(carbazol-9-yl)ethoxy)phenyl-thio]isobutyrate (ST2365)

The title product was prepared according to the procedure described inmethod B starting from methyl 2-(3-hydroxyphenylthio)isobutyrate (ST2047prepared as described in example 3) (0.609 g, 2.7 mmol),9H-carbazol-9-ethanol (0.570 g, 2.7 mmol), DIAD (0.708 g, 3.5 mmol), andtriphenylphosphine (0.917 g, 3.5 mmol) added in small portions, keepingthe temperature below 30° C., in 14 mL of anhydrous THF. The reactionmixture was left under magnetic stirring for 18 hours at roomtemperature. Then the solvent was evaporated to dryness and the residuepurified by silica gel chromatography using as eluent hexane/AcOEt 9/1.0.510 g of product were obtained (yield: 45%); Mp: 101-103° C.; TLC:silica gel, eluent hexane/AcOEt 8/2, Fr: 0.38; ¹H NMR (CDCl₃, 300 MHz) δ8.05 (d, 2H), 7.50 (m, 4H), 7.15 (m, 2H), 7.08 (t, 1H), 7.00 (d, 1H),6.90 (s, 1H), 6.80 (m, 1H), 4.75 (t, 2H), 4.35 (t, 2H), 3.60 (s, 3H),1.40 (s, 6H); HPLC: Column: Symmetry—C₁₈, (5 μm) 4.6×150 mm, R. T.,mobile phase CH₃CN/H₂O 65/35 (v/v), pH: as it is, flow rate: 0.80mL/min, 205 nm UV detector, retention time: 11.45 min; E. A. conformsfor C₂₅H₂₅NO₃S.

EXAMPLE 26 Preparation of methyl2-[4-(2-(carbazol-9-yl)ethoxy)phenyl-thio]isobutyrate (ST2387)

The product was prepared according to the procedure described in methodB starting from methyl 2-(3-hydroxyphenyl-thio)isobutyrate (ST1923prepared as described in example 1) (0.609 g, 2.7 mmol),9H-carbazol-9-ethanol (0.570 g, 2.7 mmol), DIAD (0.708 g, 3.5 mmol), towhich triphenylphosphine (0.917 g, 3.5 mmol) was added in smallportions, keeping the temperature below 30° C., in 14 mL of anhydrousTHF. The reaction mixture was left under magnetic stirring for 18 hoursat room temperature. Then the solvent was evaporated and the residuepurified by silica gel chromatography using as eluent hexane/AcOEt 9/1.0.702 g of product were obtained (yield: 62%); Mp: 72-74° C.; TLC:silica gel, eluent hexane/AcOEt 8/2, Fr: 0.30; ¹H NMR (CDCl₃, 300 MHz) δ8.05 (d, 2H), 7.50 (m, 4H), 7.15 (m, 4H), 6.75 (d, 2H), 4.75 (t, 2H),4.35 (t, 2H), 3.60 (s, 3H), 1.40 (s, 6H); HPLC: Column: Symmetry—C₁₈, (5μm) 4.6×150 mm, R. T., mobile phase CH₃CN/H₂O 70/30 (v/v), pH: as it is,flow rate: 0.80 mL/min, 205 nm UV detector, retention time: 11.60 min;E. A. conforms for C₂₅H₂₅NO₃S.

EXAMPLE 27

Constriction of the Aorta

The animals used were male Wistar rats weighing 100-120 g, housed 5 percage (cage size: 425 mm×266 mm×180 mm with sawdust litter), at atemperature of 21±1° C. and 50±15% humidity, with a light/dark cycle of12/12 h and with 15-20 air changes per hour. The animals were fed on LPALTROMIN feed (REIPER) and spring water ad libitum.

Induction of Cardiac Hypertrophy

Left ventricular hypertrophy was induced in rats anaesthetised withNembutal (pentobarbital sodium), by means of constriction of theabdominal aorta with a clip (Ø0.8 mm) placed in the abdominal aortabetween the diaphragm and the renal branches; one group of animals whichwas then used as a control group underwent the same operation but didnot have the clip implanted and therefore did not undergo constrictionof the aorta (blanks).

The animals were thus randomised to the following groups:

Blanks: operated on without constriction of the aorta (8 animals)

Controls: operated on with constriction of the aorta (8 animals)

CLO: operated on with constriction of the aorta and treated for 12 weeksfrom the day after the operation with the compounds according to theinvention described herein (11 animals).

Evaluation of Cardiac Function

At the end of the treatment cardiac function was assessed in the animalsanaesthetised with Nembutal (pentobarbital sodium), by means of apolyethylene catheter inserted in the left ventricle via the carotidartery and connected up to a pressure transducer (Statham p23XL) and toan amplifier (Biomedica Mangoni bm 61).

The parameters recorded were: heart rate, systolic and end-diastolicleft intraventricular pressure, and the positive and negativederivatives of intraventricular pressure which were recorded on apersonal computer by means of a special data acquisition system (IDAS).The recordings were carried out for 30 minutes.

Macroscopic Assessments

At the end of the experiments the animals were sacrificed by means of alethal dose of Nembutal, the abdominal cavity was opened, and theviscera were exteriorised in order to verify correct application of theaortic clip; the heart, lungs and liver were removed and, aftermacroscopic examination for possible abnormalities, were thoroughlydried and weighed.

The preliminary results obtained with this test have shown that thecompounds according to the invention described herein are well toleratedand normalise pressure values in the treated group as compared to thecontrol groups.

EXAMPLE 28

Transient Transfection of Eukaryotic Cells to Evaluate the AgonistActivity of PPARα Ligands

Transactivation assays in eukaryotic cells permit the quantitativeevaluation of the ability of a hypothetic ligand to facilitate theinteraction between a transcriptional factor and its response elementwithin a promoter.

Peroxisome Proliferator-Activated Receptor isoform alpha (PPARα)modulates target gene transcription through heterodimerization with the9-cis retinoic acid receptor (RXR). The dimer formed is capable ofbinding to the peroxisome proliferator response element (PPRE), locatedin the target gene promoter, only if activated by the presence of aligand of at least one of the two receptors

A transactivation assay thus requires the simultaneous presence in thepreselected cell line:

-   -   a) of a sufficient amount of PPARα;    -   b) of a sufficient amount of the 9 cis-retinoic acid receptor        (RXR);    -   c) of a chimeric plasmid containing the reporter gene controlled        by a PPRE, situated upstream of a heterologous viral promoter.        In our case the reporter gene is chloramphenicol-acetyl        transferase (CAT).

Whenever the endogenous levels of PPARα and RXR are insufficient, theycan be supplemented from outside sources via transfection of expressionvectors containing the genes of the receptors concerned.

The plasmid pCH110 contains the gene for β-galactosidase and isco-transfected together with the reporter gene CAT, thus providing theinternal control for transfection efficiency and normalisation of theresults.

Experimental Procedure

A cell line of monkey kidney fibroblasts (COS-7) was used. The cellswere transfected with the reporter gene (see item c above) and anexpression plasmid containing the encoding sequence of the PPARα gene(cDNA). The cells were exposed to increasing concentrations of thecompounds studied and CAT activity was assessed. Untreated cells wereused as a control. An increase in CAT levels indicates activation ofPPARα-dependent gene transcription, by means of its binding to PPRE(agonist activity of compounds).

Cell Culture

Monkey kidney fibroblasts (COS-7) were cultured according to the usualcell culture techniques at 37° C. in a 5% v/v carbon dioxide atmosphereusing as the growth medium DMEM (Dulbecco's modified Eagle's medium)modified with 3.7 g/l of sodium bicarbonate, 4 mM of L-glutamine, 4.5g/l of glucose, 1 mM of sodium pyruvate and 10% v/v of foetal bovineserum, in the presence of streptomycin 100 μg/ml and penicillin 100 U/mlfinal.

Transient Transfection of COS-7 Cells

The COS-7 cells were transiently co-transfected by means of thetechnique of co-precipitation of the nucleic acids with calciumphosphate.

The cells were plated at a density of 3×10⁵ cells/well, on plates with 6wells measuring 25 mm in diameter 24 hours prior to transfection. Theculture medium was changed 2 hours before transfection and then to eachwell were added drop-wise 280 μl of the transfection mixture prepared asfollows:

-   -   1) expression plasmid containing cDNA of PPARα (2.5 μg)    -   2) plasmid containing the reporter gene CAT (5 μg)    -   3) pCH110(1 μg);        +17.5 μl of calcium chloride 2 M.

Water was added up to a final volume of 140 μl. To this mixture ofplasmids and salt was added an equal volume of HBS solution 2×pH 7.1(sodium chloride 16 g, potassium chloride 0.74 g, basic sodium phosphatedehydrate 0.27 g, dextrose 2 g, Hepes 10 g per litre).

The cells were incubated for 6 hours at 37° C. in a 5% v/v carbondioxide atmosphere.

Treatment with the compounds according to the invention described hereinand with the reference compounds, clofibrate and 4-chloro-6-(2,3xylidino)-2-pyrimidylthioacetic acid (WY-14,643), was carried in 2 ml offresh medium for 24 h. Untreated cells were used as negative controls.The ability of the various treatments to influence the transcription ofthe reporter gene CAT was assessed radiometrically on protein extractsfrom treated and untreated cells.

Preparation of Cell Protein Extracts and Assay of CAT Activity

After the treatment, the cells were washed twice with phosphate buffer(5 ml) and removed mechanically from the wells in TEN buffer (Tris[hydroxymethyl]aminomethane 10 mM pH 8, ethylenediamine tetraacetic acid1 mM, pH 8, sodium chloride 0.1 M). After centrifuging at 4° C. for 2minutes at 1000 revs per minute (rpm) in an Eppendorf 5417R centrifuge(rotor F453011), the cells were resuspended in 0.15 ml of buffer (Tris[hydroxymethyl-aminomethane-hydrochloric acid 0.25 M, pH 8) and lysed byrepeated freezing and thawing (three 5-minute cycles).

The insoluble cell materials were removed by centrifuging at 4° C., for15 minutes at top speed and the supernatant was recovered and used forthe CAT activity assay.

The assay to measure CAT activity consists of:

-   -   1) 50 μl of protein cell extract (heated at 65° C. for 10        minutes)    -   2) 10 μl of n-butyrryl-Coenzyme A (3.5 mg/ml)    -   3) 5 μl of [¹⁴C] chloramphenicol (0.25 μCi);        in a final volume brought up to 100 μl with water.

After approximately 2 hours' incubation at 37° C. the reaction wasblocked with 2 volumes of xylene/2,6, 10,14 tetramethyl-pentadecane (ina 1:2 v/v mixture). After extraction with this solvent, 150 μl of theupper phase were added to 5 ml of scintillation liquid and analyzed witha beta-counter (scintillator) in order to determine the content of [¹⁴C]butyrryl-chloramphenicol formed as a result of the enzymatic reaction.

Test to Determine β-Galactosidase Activity

As an internal control for the normalisation of CAT activity in relationto transfection efficiency, β-galactosidase activity coded for by thecorresponding gene present in plasmid pCH110 was used.

The activity of 20 μl of protein extracts (see above) on the substrateONPG (O-nitrophenyl-β-D-galactopyranoside) 2 mg/ml was evaluated in thepresence of “Z buffer” (potassium chloride 10 mM, magnesium chloride 1mM, and β-mercaptoethanol 50 mM in phosphate buffer). After 15-120minutes' incubation at 37° C. (depending on the speed of appearance ofthe typical yellow colour), the reaction was blocked with 200 μl ofsodium carbonate 1M. The samples were incubated for 10 minutes at roomtemperature and then analyzed with a spectrophotometer, measuring theabsorbance at the wavelength of 420 nm (A₄₂₀).

The following formula was used for the normalisation of the CAT assayresults in relation to β-galactosidase activity:CAT sample count per minute−blank sample count perminute/β-galactosidase (β-gal) activity units*×CAT sample volume (50μl)/β-gal sample volume (20 μl)β-galactosidase activity units=A ₄₂₀×dilution factor/incubation time(minutes)

EXAMPLE 29

Transient Transfection of Eukaryotic Cells to evaluate the AgonistActivity of PPARα Ligands (II Method)

An alternative transactivation system, which differs mainly in the waythe receptor is positioned onto the DNA, and depending on how the eventof ligand binding is translated into transcriptional activation, wasused.

In this model eukaryotic cells were transiently transfected with anexpression vector encoding a fusion protein between the DNA bindingdomain (DBD) of the yeast Ga14 transcription factor and the ligandbinding domain (LBD) of the PPARα (Ga14DBD/PPARαLBD). The reportervector containing 5 copies of the high affinity binding site for Ga14(named UAS, upstream activating sequence) upstream of a strong viralpromoter linked to the reporter gene chloramphenicol acetyltransferase(CAT), was co-transfected. This model offered some advantages, the mostimportant of which was the absence of interference by endogenousreceptors.

Besides expression and reporter vectors, cells were transfected with acontrol vector pCH110 that encodes the β-galactosidase enzyme to correctfor differences in transfection efficiency.

Experimental Procedure

A monkey kidney fibroblast cell line (COS-7) was used. Cells wereco-transfected with the plasmid carrying the gene-reporter, theexpression plasmid encoding the fusion protein Ga14DBD/PPARαLBD, and thecontrol vector pCH110. Cells were then treated with increasingconcentrations of test compounds and the CAT activity was measured.Untreated cells were used as control.

Cell Culture

Monkey kidney fibroblasts (COS-7) were routinely grown in DMEM(Dulbecco's modified Eagle's medium) supplemented with 3.7 g/l sodiumbicarbonate, 4 mM L-glutamine, 4.5 g/l glucose, 1 mM sodium piruvate and10% v/v foetal bovine serum, in the presence of streptomycin 100 μg/mland penicillin 100 U/ml.

Transient Transfection of COS-7 Cells

COS-7 cells were transiently transfected by using the multi-componentlipid-based FuGENE6 Transfection Reagent that complexes with andtransports DNA into the cells during transfection. Cells were seeded at1.2×10⁵ cells/well, in 12-well plates, and cultured overnight at 37° C.in a 5% v/v carbon dioxide atmosphere. Two hours before transfection theculture medium was replaced by fresh serum-free medium and thentransfection was performed with FuGENE6 Transfection Reagent accordingto the instructions of the manufacturer. Briefly, the transfectionmixture containing (for each well) 0.8 μg of the expression vector, 1.6μg of the reporter vector, 0.8 μg of the control vector and 9 μl ofFuGENE6 Transfection Reagent was added directly to the cells in thepresence of serum-free medium. After 5 hours the transfection medium wasreplaced by 1 ml of the complete culture medium with or without the testmolecules at 3 different concentrations (2, 20 and 100 μM). 2 μMWy-14,643, a known PPARα ligand, was used as positive control.

Preparation of Cell Protein Extracts and Assay of CAT Activity

After 48 h, the cells were washed twice with 1 ml phosphate buffer (PBS)and then harvested by scraping them in TEN buffer (Tris[hydroxymethyl]aminomethane 10 mM pH 8, ethylenediamine tetraacetic acid1 mM, pH 8, sodium chloride 0.1 M). Following centrifugation at roomtemperature, for 3 min at 1000 revs per minute (rpm), cells wereresuspended with 60 μl of Lysis buffer (0.25M Tris-HCl, pH 8) and lysedby three rapid freeze/thaw cycles (three 5-minute cycles). Cell debriswas then removed by centrifugating at 4° C., for 15 min at 15.000 revsper minute (rpm),. Glycerol (final 10% v/v) and β-mercaptoethanol (final5 mM) were then added (final volume 75 μl) and the cell extracts werestored at −80° C. until assayed.

The CAT activity assay was performed as follows: 20 μl of cell lysate(prewarmed at 65° C. for 10 min to deactivate internal deacetylaseenzymatic activity) were added to 10 μl of 3.5 mg/ml n-butirryl-CoA, 5μl (0.25 μCi) of [¹⁴C]-chloramphenicol and 65 μl of distilled H₂O andincubated 2 h at 37° C. Reaction was blocked by adding 200 μl of thesolution xylene/2,6,10,14 tetramethyl-pentadecane (in a 1:2 v/vmixture). After a vigorous vortexing and centrifugation for 5 min at topspeed, 150 μl of supernatant were transferred to scintillation vial inthe presence of 5 ml of scintillation liquid, and the relativeradioactivity was measured by a β-counter.

Test to Determine β-Galactosidase Activity

The β-galactosidase activity was measured as follows: 20 μl of cellularextracts were added to 750 μl of reaction buffer consisting of 1 volumeof 2 mg/ml ONPG and 3 volumes of “Z buffer” (potassium chloride 10 mM,magnesium chloride 1 mM, and β-mercaptoethanol 50 mM in phosphatebuffer). Reaction was performed at 37° C. and blocked by adding 200 μlof 1M Na₂CO₃ when a typical yellow colour became appreciable. Sampleswere incubated for 10 min at room temperature and then the absorbance at420 nm (A₄₂₀) was spectrophotometrically measured.

The CAT activity results were normalized to the β-galactosidase activityas follows: $\begin{matrix}{A_{420}\quad x\quad{dilution}\quad{factor}} \\{\beta - {{galactosidase}\quad{units}*}}\end{matrix} = \frac{\frac{{{CAT}\quad{sample}\quad{count}\quad{per}\quad{minute}} - {{blank}\quad{sample}\quad{count}\quad{per}\quad{minute}}}{\beta - {{galactosidase}\quad{activity}\quad{units}*}}}{{incubation}\quad{time}\quad\left( \min \right)}$

The preliminary results obtained, reported in Table 1, show that thecompounds according to the invention are PPARα agonists. TABLE 1Compound 2 μM 20 μM 100 μM Example 5 (ST1983) 150% 391, 2% 1372% Example14 (ST2167)  98, 1% 360% 462, 7% Example 24 (ST2534) 113, 1% 284, 9% 421%

The results are expressed as percentage activation of the CAT reportergene compared to that measured in the presence of the reference compound(WY-14.643 2 μM), conventionally taken as equal to 100%.

EXAMPLE 30

Increase in HDL-Cholesterol Levels in db/db Mice

In this experiment db/db mice were used in which PPARα expression isabove normal (Memon et al., Endocrinology 2000, 4021-4031) andHDL-cholesterol levels are substantially elevated (Silver et al., J BiolChem 1999, 274: 4140-4146).

The C57BL/KsJ db/db mice were acclimatised for one week in standardconditions (22±2° C.; 55±15% humidity; 15-20 air changes/hour; 12 hourslight/darkness cycle with light from 7.00 a.m. to 7 p.m.) with astandard 4 RF21 diet (Mucedola). Blood samples were taken inpost-absorption conditions (fasting from 8.30 a.m. to 4.30 p.m.) fromthe caudal vein with the aid of a Jelco 22G catheter (Johnson andJohnson). Glucose, insulin, triglyceride, cholesterol, free fatty acidand urea levels were checked in plasma for a homogeneous distribution ofthe mice in the treatment groups.

At the beginning of treatment the animals' body weight was checked andarrangements were made to monitor their water and feed consumption.

The mice were treated twice daily (at 8.30 a.m. and 6.30 p.m.) orallyfor 10 or 14 days.

The compound tested, obtained as described in example 4 (ST 1929) wasadministered at the dose of 24 mg/kg in 10 ml/kg of vehicle (1% CMCcontaining Tween 80 0.5% in deionized H₂O).

The other compounds tested were also administered at a dose equivalentto that in example 4.

Ciprofibrate, a known PPARα agonist (Varanasi et al., J Biol Chem 1996,271: 2147-2155; Latruffe et al. Cell Biochem Biophys 2000, 32 Spring:213-220) was administered at the dose of 20 mg/kg (Dwivedi et al.,Toxicol Pathol 1989, 17: 16-26; Qi et al., Proc Natl Acad Sci USA 1999,96: 1585-1590).

The animals were sacrificed (by decapitation) in conditions ofpost-absorption (fasting from 9.30 a.m. to 4.30 p.m.) 7 hours after thelast treatment. The levels of a number of important lipid andcarbohydrate metabolism parameters were determined in the serum.

The HDL-cholesterol levels were measured by treating the serum withphosphotungstic-acid-based precipitating reagent (ABX Diagnostics) whichremoves the chylomicrons, very low density and low density lipoproteinsand determining the HDL-cholesterol levels in the supernatant with theaid of the Cholesterol Kit (ABX Diagnostics) and the Cobas Mira SAutoanalyzer (Roche).

The results indicate that, in db/db mice, the compounds according to theinvention are capable of raising HDL-cholesterol values (indicator ofPPARα agonist activity) in a manner similar to or greater than thereference compound, ciprofibrate (Table 2). TABLE 2 Increase inHDL-cholesterol levels in db/db mice Increase in Duration ofHDL-cholesterol Dose treatment levels Compound mg/kg (days) (%)Ciprofibrate 20 14 +52 ▴ Example 4 24 10 +80 ▴ Compound (ST1929) Example8 Equivalent to 10 +51 ▴ Compound 24 mg of (ST2031) ST1929Student's ‘t’-test: ▴ indicates P < 0.001 vs control.

The compounds of formula (I) according to the invention described hereincan be used as such or in the form of pharmaceutically acceptablederivatives, such as salts, or derivatives that improve thepharmacokinetic aspects, while maintaining the specific activity(prodrugs).

As far as the industrial aspect of the invention described herein isconcerned, the medicines will be in the form of suitable pharmaceuticalformulations (or compositions), prepared according to conventionalmethods with which the expert in the sector is familiar. Examples ofpharmaceutical compositions are tablets, capsules, pills, suppositories,sachets, liquid forms for oral administration, such as solutions,suspensions and emulsions; controlled release forms for oral or enteraladministration in general; and forms for parenteral administration, suchas injectable forms.

1. Formula (I) compounds

in which: R represents —H, —YCR5R6COX, monocyclic, bicyclic or tricyclicaryl or heteroaryl (possibly substituted by groups of the type—YCR5R6COX, halogens, nitro, hydroxy, alkyl, and alkoxy, possiblysubstituted by halogens), monocyclic, bicyclic or tricyclic arylalkyl orheteroarylalkyl (in which the aryl or heteroaryl are possiblysubstituted by groups of the type —YCR5R6COX, halogens, nitro, hydroxy,alkyl, and alkoxy, possibly substituted by halogens). The heteroaryl maypossibly be charged, of the type:

in which the positive charge is balanced by a suitable negativecounterion; m represents 0-1 n represents 0-3; when n represents 1, R3and R4, which may be the same or different, are selected from H or alkylC₁-C₅; when n represents 2 or 3, R3 is equal to R4 and represents H; prepresents 0-1 X represents —OH, —O-alkyl C₁-C₃; R1 and R2, which may bethe same or different, are selected from: —H; alkyl C₁-C₅; -alkoxy,possibly substituted by halogens; -phenoxy, possibly substituted byhalogens, nitro, hydroxy, alkyls; -benzyloxy, possibly substituted byhalogens, nitro, hydroxy, alkyls; —COX; or together with COX of generalformula (I) form a cycle of the type:

R5 and R6, which may be the same or different, are selected from thegroups listed for R1 and R2; Q and Z, which may be the same ordifferent, are selected from: NH, O, S, —NHC(O)O—, NHC(O)NH—, —NHC(O)S—,—OC(O)NH—, —NHC(S)O—, —NHC(S)NH—, —C(O)NH—; and Y represents O, S. 2.Formula (I) compounds according to claim 1, for use in the medicalfield.
 3. Pharmaceutical composition containing as its active ingredienta formula (a) compound according to claim 1 and at least onepharmaceutically acceptable excipient and/or diluent.
 4. Compositionaccording to claim 3, in the form of tablets, capsules, pills, sachets,vials, powders, suppositories, solutions, suspensions, emulsions orliposomal preparations.
 5. Composition according to claim 4, which canbe administered by the enteral or parenteral routes.
 6. Use of formula(I) compounds according to claim 1 for the preparation of a medicine forthe treatment of diseases responding to PPARα activation.
 7. Useaccording to claim 6, in which the diseases are selected from the groupconsisting of heart failure, the hyperlipaemias and atherosclerosis.